Infection with the high risk types of human papillomaviruses (HPVs) is strongly linked to the development of cancers of the uterine cervix. Carcinogenesis depends upon the continuous expression of the viral E6 and E7 oncogenes in the affected individual. Transcription of these oncogenes is negatively regulated by, the viral E2 protein. A frequent characteristic of carcinogenic progression of HPV positive lesions is the integration of the viral DNA into the cellular genome, accompanied by the disruption of the viral E2 open reading frame. When reintroduced into HPV positive cancer cells, E2 proteins suppress cellular growth through senescence induction. E2 mediated repression of E6/E7 expression is both necessary and sufficient for this process, indicating that important senescence mediators must be inhibited by the viral oncoproteins. This proposal aims to investigate the targeting of the cellular senescence machinery by HPV oncoproteins and to uncover basic molecular relationships between HPV carcinogenesis and cellular senescence. Our studies are interfaced with an assessment of the clinical potential that E2 may have in the treatment of HPV associated lesions. Using an E2 based inducible system, we have monitored the transcriptome of HPV positive cells during an early, yet irreversibly committed senescence stage. By detecting message levels of candidate genes in HPV positive versus HPV negative cells, we will first identify senescence associated genes, which are targeted by E6 or E7. Transcriptional regulation of these genes will be investigated in response to various wild type and mutant E6 and E7 proteins. The results will i) yield insights into mechanisms of gene regulation by E6 and E7 and ii) correlate immortalizing oncogene activities with the targeting of specific senescence-associated molecules. Monitoring the regulation of candidate genes during senescence in vitro will be extended to functional studies. We will utilize RNA interference to probe the role of genes that are unregulated during senescence. Similarly, we will overexpress molecules that are repressed during senescence. Regulators of the balance between senescence and transformation as identified in these studies may represent novel drug targets and diagnostic markers for the inhibition of tumorigenesis in humans.